1. Field of the Invention
The present invention generally relates to multidimensional electrophoresis devices and methods of making and using thereof.
2. Description of the Related Art
The advent of microfluidic chips has enabled miniaturization of many biochemical techniques resulting in faster and cheaper analysis using much smaller amounts of sample and reagents. Microfluidic devices have in many ways revolutionized the analytical capabilities available for chemistry, biology, and medicine. Microfluidic devices allow analysis using minute amounts of samples (crucial when analyzing body fluids or expensive drug formulations), are fast, and enable development of portable systems. One of the biggest advantages offered by microfluidic chips, analogous to microelectronics chips, is the potential for seamless integration of functions at the chip-scale.
While great advances have been made in integrating some functions such as injection and analysis; in most cases sample pretreatment is performed off-chip. Recently, approaches have been developed to incorporate functions such as sample cleanup, sample concentration, mixing, and reaction prior to analysis in microchips. See Auroux et al. (2002) Anal. Chem. 74:2637; Reyes et al. (2002) Anal. Chem. 74:2623; and Vilkner et al. (2004) Anal. Chem. 76:3373.
There are a number of reasons why sample concentration prior to analysis is a crucial step in development of multi-functional integrated microfluidic devices. For example, preconcentration of sample enables detection of trace or low-abundant species. This is of particular importance in many fields including clinical diagnostics, proteomics, forensics, environmental monitoring and biodefense applications. Also, micrometer dimensions of the fluidic channels lead to poorer sensitivities for optical detection than their conventional scale counterparts. Preconcentration not only improves detection sensitivity but also improves the reliability of analysis by significantly increasing signal-to-noise ratios. Further, the practical constraints on sample loading limit the minimum volume of sample inserted into a chip to the order of about 1 μl while the volume typically analyzed is on the order of about 1 nl. Hence, analytes in a sample can be concentrated up to 1000-fold without requiring additional sample.
Reported sample preconcentration methods can be categorized into many groups including surface-binding, electrokinetic equilibrium, and porous membrane techniques. Surface-binding techniques such as solid phase extraction or affinity columns use sample adsorption to surfaces for concentration and a solvent or surface property change for elution. See Broyles et al. (2003) Anal. Chem. 75:2761; Jemere et al. (2002) J. Electrophoresis 23:3537; and Yu et al. (2001) Anal. Chem. 73:5088. Electrokinetic equilibrium techniques concentrate sample by bringing species transport to a local equilibrium state electrokinetically and examples include isoelectric focusing (IEF) and field amplified sample stacking (FASS) or isotachophoresis (ITP). See Li et al. (2003) J. Electrophoresis 24:193; Cabrera & Yager (2002) J. Electrophoresis 22:355; Huang & Pawliszyn (2002) J. Electrophoresis 23:3504; and Wainright et al. (2002) J. Chromatography A 69:979.
Approaches have also been developed that rely on the concept of size-based exclusion to concentrate macromolecules using a porous membrane that excludes species of interest from the membrane pores. See Foote et al. (2005) Anal Chem. 77:57; Song et al. (2004) Anal Chem. 76:4589; Khandurina et al. (1999) Anal. Chem. 71:1815; and U.S. Publication No. 20040084370. Each of these approaches has its own advantages and drawbacks. For example, sample stacking methods require insertion and maintenance of multiple buffer zones and can be difficult to implement with samples of unknown conductivity. Affinity-based preconcentration requires a change in buffer conditions for elution. A size-exclusion or filtration-based approach is arguably the easiest to implement as it avoids complications of specifically arranging zones of buffer and reagents or the need for selective binding and release of analytes while offering high sample capacities with concentration factors greater than 1,000-fold.
However, a size-exclusion or filtration-based approach requires placement of nanoporous membranes or filters inside specific channels. Khandurina et al. demonstrated a size-exclusion approach for concentrating DNA and more recently for concentrating proteins, wherein a silicate membrane was deposited between the glass cover plate and silicon substrate of a microchip. See Khandurina et al. (1999) Anal. Chem. 71:1815. While a 600-fold signal increase was reported for proteins electrophoretically driven against the silicate membrane, the authors reported that (1) the chips are hard to fabricate in a reproducible manner, and (2) the silicate membrane often has defects adversely affecting the concentration. The requirement on fabrication is that channels bridged by the silicate membrane must be etched such that their edges are separated by a few microns. This required precision can be a demanding and limiting fabrication requirement, especially when etching deep channels with an isotropic etch process. Another limitation is that the surface area of the membrane face is a very thin line of contact between the channel lid and the top of the channel wall. The maximum flux and trapping area are dependent on the surface area of the face.
Recently, Wang et al. reported a novel preconcentrator approach with up to million-fold concentration of proteins and peptides using a nanofluidic filter that requires fabrication of micro- and nano-channels in the same chip. See Wang et al. (2005) Anal. Chem. 77:4293-4299. The nanofluidic filter described by Wang et al. requires labor intensive and demanding fabrication process to make the nanometer size channels. There are other aspects of the nanofilter that pose difficulties for integrated processing and analysis. For example, the trapping mechanism relies not on size-exclusion to trap species, but on generating an ion depletion zone that begins trapping all charged species. This means there is no option for size-selective trapping or filtering and that small buffer ions are stacked along with larger analyte which can be detrimental for downstream processing, analysis or both, e.g. if the concentrated species were directed into a gel electrophoresis channel, sample destacking would prohibit useful separations.
Further, non-linear concentration factors and lack of reproducibility are problematic with a membrane based approach to preconcentration. See Foote et al. (2005) Anal. Chem. 77:57. This behavior results from concentration polarization that can lead to sample destacking. In the pores of a size-exclusion membrane the thickness of the electrical double layer (EDL) can be on the same order of magnitude as the pore radius. For a negatively-charged membrane such as glass or (and to a lesser degree) polyacrylamide, this results in selective enrichment of cations in the pores. In the absence of an applied electric field, a boundary potential (Donnan potential) exists between bulk and the membrane to equalize the concentrations of ions. When an applied electric field is superimposed, concentration polarization results where concentrations of ions increases on the cathodic side and decreases on the anodic side. The steep concentration gradients in the depleted concentration polarization zone results in diffusion-limited transport of ions. This leads to a drop in current as a function of time upon application of the external electric field. At the diffusion limit, the current reaches a steady value referred to as “limiting current”. Localized increase in ion concentrations lead to sample destacking and other non-linear effects resulting in band-broadening and irreproducible migration over time.
Thus, a need exists for methods and devices for preconcentrating a sample on a microchip that are readily and easily fabricated, produce consistent results and do not suffer from concentration polarization problems.